We are working to establish an in vitro system for the study of checkpoint activation by various agents that induce DNA damage in human cells. Some of these DNA damaging agents are widely used in chemotherapy. Thus, our research focuses on molecular mechanisms by which these chemotherapeutic agents confer cytotoxicity as well as pathways by which cancer cells achieve tolerance. As a model system, we are investigating checkpoint activation mediated by the DNA mismatch repair proteins MutSalpha and MutLalpha in response to several DNA damaging agents including SN1 alkylators, fluorouracil, and cisplatin. The cellular response to DNA damage involves the activation of signaling kinases such as ATR and ATM. This, in turn, activates a cascade of signaling kinases including Chk1 and Chk2 that ultimately result in cell cycle arrest in S phase and at the G2/M boundary. We are purifying recombinant human proteins involved in this damage response with the goal of defining the underlying molecular mechanism. We are identifying components of key protein complexes that function to license cell cycle arrest in response to DNA damaging agents. This work involves a collaboration with Dr. Dorothy Erie, UNC-Chapel Hill, to use single molecule imaging techniques to characterize protein complexes. We are also examining the role of nuclear membrane proteins in coordinating dynamic changes in nuclear architecture during the cell cycle and in response to DNA damage. We are identifying sites of cell cycle-dependent modification of components of the nuclear pore complex by mass spectroscopy in collaboration with the NIDDK Mass Spectroscopy Core facility. We are using a variety of functional assays to define the roles that these modifications play in response to signals for cell division and DNA damage.